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US 20020090376A1
(19)
United States
(12)
Patent Application Publication
(lo)
Pub. No.:
US 2002/0090376 Al
KANIGA et at.
(43)Pub. Date:
Jul. 11, 2002
(54) METHODS OF PRODUCING AND USING VIRULENCE ATTENUATED PDXR MUTANT BACTERIA
(76) Inventors: KONE KANIGA, ST. LOUIS, MO (US); PREETI SUNDARAM, CHESTERFIELD, MO (US)
Correspondence Address: THOMPSON COBURN, LLP ONE FIRSTAR PLAZA SUITE 3500
ST LOUIS, MO 63101 (US)
(*) Notice: This is a publication of a continued pros- ecution application (CPA) filed under 37 CFR 1.53(d).
(21) Appl. No.: 08/829,402 (22) Filed: Mar. 31, 1997
Publication Classification
(51) Int. Cl.7 ... A61K 39/00; A61K 39/38 (52) U.S. Cl . ... 424/184.1
(57) ABSTRACT
Disclosed are bacteria having virulence attenuated by a mutation to the regulatory gene poxR. Also disclosed is a method of producing bacteria having virulence attenuated by mutating to the regulatory gene poxR. Such bacteria are useful for inducing an immune response in an animal or human against virulent forms of the bacteria with reduced risk of a virulent infection. Such bacteria are also useful to allow use of normally virulent bacteria as research tools with reduced risk of virulent infection. In a preferred embodi-ment, poxR attenuated bacteria can be used as a vaccine to induce immunoprotection in an animal against virulent forms of the bacteria. The disclosed bacteria can also be used as hosts for the expression of heterologous genes and proteins or to deliver DNA for genetic immunization. Attenuated bacteria with such expression can be used, for example, to deliver and present heterologous antigens to the immune system of an animal. Such presentation on live bacteria can lead to improved stimulation of an immune response by the animal to the antigens. It has been discov-ered that bacteria harboring a poxR mutation has signifi-cantly reduced virulence. Also disclosed is the nucleotide sequence of the poxR gene from Salmonella typhimurium, and the amino acid sequence of the encoded protein. The encoded protein has 325 amino acids and has significant sequence similarity to previously uncharacterized open reading frames in E. coli and Haemophilus influenzae.
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Sau3AI GATCGGCTTGAAAGGTTTGCACGACATTCCTCCAGATTATTGTAATTTCACCCTCGCGCAGCCAGATAAAGCCTCTGGGT TCTGCGAAGTATGAATGCGTTTCCACTGCTCCTTTATGGGTACAACAGTATAGTCTCAGGGATGTGAGGGAAATTTGACC TGTTCGATTTTTTTAGCGTATCAGAGGGATGAATTATCATTGATTTTGATTAATTTAATTACTAAACCATCTGA,-k TCAC TTTTTTTACCCTCCAGAAGGCGCCCGATACGCCTGCGCAAP_ ,TTTGTTTCGCCCGCGCGTTGCGAGTAGACTTCGTGACC 345/1 375/11TTGTCTTAACTGGAGAAAGAR'_^C ATG AGC GAA ACG GCA ACC TGG CAG CCG AGC GCG TCC AT Q CCC SD M S E T A T W Q P S A S I P
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Patent Application Publication Jul. 11, 2002 Sheet 7 of 7
US 2002/0090376 Al
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GCA CGT GAG CAA CAA CAG CGC TTT GAA CAG GAC AT CGT AAG CGC GCC GCT CGC GGT CTG A R £ Q Q Q R F E Q D N R K R A A R G L
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-TT _C._TOG TAT GTC ATA. TGG ATC- GTA AGT ACG GCG GCA AA ATT TGG GTA CCG TTT TCT
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US 2002/0090376 Al
Jul. 11, 2002
METHODS OF PRODUCING AND USING VIRULENCE ATTENUATED PDXR MUTANT
BACTERIA
BACKGROUND OF THE INVENTION [0001] The disclosed invention is in the general areas of bacteria with attenuated virulence and live bacterial vac-cines.
[0002] The poxA gene (Van Dyk et al., J. Bacteriology 169(10):4540-4546 (1987)), is a regulatory gene affecting expression of pyruvate oxidase (Chang and Cronan, J. Bacteriology 151(3):1279-1289 (1982)). The poxA gene of E. coli is located at min 94. Enzymological and immuno-logical data indicate that mutations in poxA have an 8 to 10-fold decrease in pyruvate oxidase levels (Chang and Cronan (1982); Chang and Cronan, J. Bacteriol. 154:756-762 (1983)). It has also been reported that poxA mutants grow more slowly than the isogenic wild-type in both minimal and rich media, while poxB mutants exhibit normal growth. Van Dyk and LaRossa, J. Bacteriol. 165(2):386-392 (1986), isolated 15 mutant Salmonella typhimurium strains sensitive to the herbicide sulforneturon methyl (SM) [N-[(4, 6-dimethylpyrimidin-2-yl)aminocarbonyl]-2-methoxycar-bonyl-benzenesulfonamide], following Tn10 mutagenesis. Among these SM-hypersensitive mutations, a poxA muta-tion was identified and mapped to the 94 min region of S. typhimurium genetic map (Van Dyk et al. (1987)), a location analogous to that of poxA in E. coli. The S. typhimurium poxA mutant, similarly to the E. coli poxA mutant, had reduced pyruvate oxidase activity and reduced growth rates (Van Dyk et al. (1987)). Furthermore, the E. coli and S. typhimurium poxA mutants shared several additional phe-notypes including hypersensitivity to SM, to a-ketobutyrate, and to a wide range of bacterial growth inhibitors, such as antibiotics, amino acid analogs and dyes (Van Dyk et al. (1987)).
[0003] The immune system of animals is especially suited to reacting to and eliminating microorganisms which infect the animal. The sustained presence of the full range of antigens expressed by infecting microorganisms provide a stimulating target for the immune system. It is likely that these characteristics lead to the superior efficacy, on average, of vaccines using live attenuated virus. For similar reasons, live bacterial vaccines have been developed that express a desired antigen and colonize the intestinal tract of animals (Curtiss et al., Curr Topics Micro. Immun. 146:35-49 (1989); Curtiss, Attenuated Salmonella Strains as Live Vec-tors for the Expression of Foreign Antigens, in New Gen-eration Vaccines (Woodrow and Levine, eds., Marcel Dek-ker, New York, 1990) pages 161-188; Schodel, Infection 20(1):1-8 (1992); Cardenas and Elements, Clinical Micro. Rev. 5(3):328-342 (1992)). Most work to date has used avirulent Salmonella typhimurium strains synthesizing vari-ous foreign antigens for immunization of mice, chickens and pigs. Several avirulent S. typhi vectors have been evaluated in human volunteers (Tacket et al., Infect. Immun. 60:536-541 (1992)) and several phase I clinical trials with recom-binant avirulent S. typhi strains are in progress in the U.S. and Europe. An important safety advantage of the live attenuated bacterial vaccine vectors as compared to the use of viral vector based vaccines is the ability to treat an immunized patient with oral ciprofloxacin or amoxicillin, should an adverse reaction occur.
[0004] It is understood that live bacteria administered to animals, and especially to humans, should not be patho-genic. Thus, live bacterial vaccines need to be avirulent. However, many bacteria that are most effective in stimulat-ing an immune response, and would thus be most attractive as the subject of a live bacterial vaccine, are also the most virulent. Accordingly, there is a need for methods to reduce, or attenuate, the virulence of bacteria.
[0005] It is therefore an object of the present invention to provide a method of attenuating the virulence of a bacteria. [0006] It is another object of the present invention to provide a method for inducing an immune response in an animal using bacterial cells with attenuated virulence. [0007] It is another object of the present invention to provide compositions for inducing an immune response in an animal comprising bacterial cells with attenuated viru-lence.
[0008] It is another object of the present invention to provide an isolated nucleic acid molecule containing or encoding the poxR gene.
BRIEF SUMMARY OF THE INVENTION [0009] Disclosed are bacteria having virulence attenuated by a mutation to the regulatory gene poxR. Also disclosed is a method of producing bacteria having virulence attenuated by mutating the regulatory gene poxR. Such bacteria are useful for inducing an immune response in an animal or human against virulent forms of the bacteria with reduced risk of a virulent infection. Such bacteria are also useful to allow use of normally virulent bacteria as research tools with reduced risk of virulent infection. In a preferred embodi-ment, poxR attenuated bacteria can be used as a vaccine to induce immunoprotection in an animal or human against virulent forms of the bacteria. The disclosed bacteria can also be used as hosts for the expression of heterologous genes and proteins. Attenuated bacteria with such expression can be used, for example, to deliver and present heterolo-gous antigens to the immune system of an animal. Such presentation on live bacteria can lead to improved stimula-tion of an immune response by the animal to the antigens. [0010] The poxR gene, which is referred to in the literature as poxA (Van Dyk et al., J. Bacteriology 169(10):4540-4546 (1987)), is a regulatory gene affecting expression of pyru-vate oxidase (Chang and Cronan, J. Bacteriology 151(3):1279-1289 (1982)) and having other, pleiotropic effects. The pleiotropic phenotype effects include reduced pyruvate oxidase activity, reduced growth rate, hypersensi-tivity to the herbicide sulfometuron methyl, to a-ketobu-tyrate and to amino acid analogs, and failure to grow in the presence of the host antimicrobial peptide, protamine. It has now been discovered that bacterial cell harboring a poxR mutation has significantly reduced virulence. Also disclosed is the nucleotide sequence of the poxR gene from Salmo-nella typhimurium, and the amino acid sequence of the encoded protein. The encoded protein has 325 amino acids and has significant sequence similarity to previously unchar-acterized open reading frames in E. coli and Haemophilus influenzae.
BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 is a graph of the number of poxR mutant S. typhimurium UK-1 cells colonizing various organs (mea-
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2
sured in colony forming units per gram of organ) in day-old chicks. Chicks were orally inoculated with 1x10 8 cfu of poxR401::Tn10 strain MGN-791s at day of hatch. All birds survived and six days post-infection, the colonization levels in the spleen, liver, bursa and cecum were determined. [0012] FIG. 2 is a graph of the number of poxR mutant S. typhimurium cells colonizing various organs (measured in mean colony forming units per organ) in BALB/c mice. Each time point consisted of group of three female BALB/c mice six weeks-old. Mice were inoculated orally (p.o.) with 2.6x109 cfu at day 1. Three and seven days post-inoculation, mice were humanely euthanized and spleens (SP), mesen-teric lymph nodes (MLN), and Peyer's patches (PP) were removed. The organs were disrupted in one milliliter of buffer saline and 0.1 ml of appropriate dilutions were plated in triplicate on MacConkey lactose plates.
[0013] FIGS. 3A, 3B, and 3C are graphs of the immune responses (measured as optical density at 405 nm of ELISA assays) of mice immunized with various amounts of S. typhimurium poxR mutant. The optical density at 405 nm in the ELISA assays is a measure of the level of immunore-active antibodies present.
[0014] FIG. 3A graphs the IgA response. [0015] FIG. 3B graphs the IgM response.
[0016] FIG. 3C graphs the IgG response. Each treatment group consisted of five female BALB/c mice six weeks-old. Mice were inoculated intra peritoneally (i.p.) with 2.6x10 4 cfu, and orally (p.o.) with 2.6x10 7, 2.6x108, and 2.6x109 cfu at day 1. No booster immunization was performed. At day 28 post-immunization, sera were collected from immunized and non immunized control mice and subjected to an ELISA assay using purified Salmonella LPS as coating antigen. The graphs represent the mean absorbance at 405 nm (OD) for five mice. For individual mice, the serum was considered positive when the OD was greater than the mean OD of control mice plus two times the standard deviation of the same control sera. For the IgA response, 5/5 were positive in the i.p. group, 3/5 in the p.o. 2.6x10 7 cfu group; 515 in the p.o. 2.6x108 cfu group; 3/5 in the p.o. 2.6x10 9 cfu group. For the IgM response, 3/5 were positive in the i.p. group; 2/5 in the p.o. 2.6x107 cfu group; 2/5 in the p.o. 2.6x10 8 cfu group; 2/5 in the p.o. 2.6x10 9 cfu group. For the IgG response, 5/5 were positive in the i.p. group; 4/5 in the p.o. 2.6x10 7 cfu group; '5/5 in the p.o. 2.6x10 8 cfu group; 3/5 in the p.o. 2.6x10 cfu group.
[0017] FIG. 4 is a diagram of partial restriction endonu-clease maps of the poxR insert of plasmids pMEG-250, pMEG-251, pMEG-273, pMEG-280, and pMEG-274. The position of cleavage sites for some restriction endonucleases are shown. The direction of transcription of the poxR gene is indicated by the arrow. The plasmids from which each plasmid was derived is shown in parentheses underneath the plasmid name.
[0018] FIG. 5 is a graph of the effect of poxR mutation on invF and sipC gene expression. The expression was mea-sured in units of catechol 2,3 dioxygenase, the product of the xylE reporter gene which was fused, in separate strains, to the chromosomal invF, sipC and asd genes. The poxR mutation was introduced in these strains by P22 transduction as described in the Example 4. Units are expressed as picomoles of catechol 2,3 dioxygenase per mg of protein X
102.
[0019] FIGS. 6A and 6B show the nucleotide sequence of poxR gene (SEQ ID NO:1) and the deduced amino acid sequence of the encoded PoxR protein (SEQ ID NO:2). The deduced amino acid sequence encoded by a partial second open reading frame (ORF2) is also shown (SEQ ID NO:3). The nucleotide sequence starts with the Sau 3AI site and ends at the Bam HI site. The putative Shine-Dalgarno sequences upstream of the open reading frames are at nucleotides 334-338 for PoxR and nucleotides 1536-1540 for ORF2. The predicted ATG start codons begin at nucle-otide 345 for PoxR and nuclenucle-otide 1548 for ORF2.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Disclosed are bacteria having virulence attenuated by a mutation to the regulatory gene poxR. Also disclosed is a method of producing bacteria having virulence attenuated by mutating to the regulatory gene poxR. Such bacteria are useful for inducing an immune response in an animal against virulent forms of the bacteria with reduced risk of a virulent infection. Such bacteria are also useful to allow use of normally virulent bacteria as research tools with reduced risk of virulent infection. In a preferred embodiment, poxR attenuated bacteria can be used as a vaccine to induce immunoprotection in an animal or human against virulent forms of the bacteria. The disclosed bacteria can also be used as hosts for the expression of heterologous genes and proteins. Attenuated bacteria with such expression can be used, for example, to deliver and present heterologous antigens to the immune system of an animal or human. Such presentation on live bacteria can lead to improved stimula-tion of an immune response by the animal to the antigens. [0021] The poxR gene, which is referred to in the literature as poxA (Van Dyk et al. (1987)), is a regulatory gene affecting expression of pyruvate oxidase (Chang and Cronan (1982)). Pyruvate oxidase itself is encoded by the gene poxB (Chang and Cronan (1983)). Because mutants defective in poxB did not exhibit most of the various phenotypes observed in poxA mutants, we concluded that poxR is a regulatory gene having effects other than those resulting from a decrease in poxB expression. The regulatory nature of poxA leads us to rename the gene poxR.
[0022] The pyruvate oxidase of Escherichia coli is a peripheral membrane protein that catalyses the oxidative decarboxylation of pyruvate to acetate and CO 2 (Gennis and Hager, The enzymes of biological membranes, Volume 2 (Martonosi, ed., New York, N.Y., 1976), pages 493-504). Under laboratory conditions, this enzyme is not essential and conversion of pyruvate to acetate is considered wasteful of energy, compared with its conversion to acetyl coenzyme A (Gennis and Stewart, Escherichia coli and Salmonella, Volume 1 (Neidhardt, ed., ASM Press, Washington, D.C., 1996), pages 217-261). Pyruvate oxidase has been of interest primarily as a model for studying protein-lipid interaction. The enzyme is a water-soluble tetramer of 62 kDa identical subunits (Gennis and Stewart, 1996). It requires thiamine pyrophosphate, flavin adenine dinucleotide, and Mg t as cofactors (Gennis and Hager (1976); Bertagnolli and Hager, J. Biol. Chem. 266:10168-10173 (1991); Bertagnolli and Hager, Arch. Biochem. Biophys. 300:364-371 (1993)). In the presence of the substrate and cofactors, the enzyme under-goes conformational changes and binds to E. coli membrane vesicles and to phospholipid vesicles (Russell et al., J. Biol.
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3
Chem. 252:7883-7887 (1977); Schrock and Gennis, Bio-chim. Biophys. Acta 614:215-220 (1980)). This peripheral membrane binding is necessary for the terminal transfer of electron to ubiquinone-8 which is dissolved in the lipid bilayer (Grabau and Cronan, Biochemistry 25:3748-3751 (1986); Koland et al., Biochemistry 23:445-453 (1984)). [0023] The poxR gene has other regulatory effects as evidenced by the pleiotropic phenotype of poxR mutants. Observed effects include reduced pyruvate oxidase activity, reduced growth rate, hypersensitivity to the herbicide sul-fometuron methyl, to a-ketobutyrate and to amino acid analogs, and failure to grow in the presence of the host antimicrobial peptide, protamine. The structural gene for pyruvate oxidase, poxB, has been located at min 18.7 on the E. coli genetic map (Chang and Cronan (1983)).
[0024] A. poxR Genes
[0025] The poxR gene of Salmonella typhimurium was cloned as described in Example 1. The disclosed poxR gene (SEQ ID NO:1), and nucleic acids derived from the poxR
gene, can be used in the disclosed methods to mutate the
poxR gene in a bacterial cell. The poxR gene, and nucleic acids derived from the poxR gene, can also be used to identify, map, and clone other poxR genes and genes homologous to poxR. The poxR gene, and nucleic acids derived from the poxR gene, can also be used to determine the structure of poxR mutants by, for example, Southern blotting or Northern blotting. The poxR gene can also be used to produce PoxR protein, using, for example, recom-binant DNA expression techniques. Nucleic acids derived from the poxR gene can also be used as specific probes or primers for use in, for example, the polymerase chain reaction (PCR) or related amplification methods, nucleic acid sequencing, detection of the presence or absence of specific poxR sequences in nucleic acid samples. These and many other techniques for the general use of nucleotide and gene sequences are known and can be practiced using the disclosed poxR gene and nucleic acids derived from the
poxR gene. It is preferred that the disclosed poxR gene be used to mutate the poxR genes of bacterial cells and to clone other poxR genes. An example of the use of the disclosed
poxR gene for mutating the poxR gene in a bacterial cell is described in Example 3.
[0026] The PoxR protein (SEQ ID NO:2) encoded by the
poxR gene can be used to produce antibodies immunoreac-tive to the PoxR protein. Such antibodies can be used, for example, to identify or detect PoxR protein, peptides derived from the PoxR protein, or proteins related to the PoxR
protein, in a method such as Western blotting. Such anti-bodies can also be used to clone, or to identify clones, of
poxR, genes homologous to poxR, or other related genes. Methods for the production of antibodies, and numerous techniques for their use, are known. Numerous examples of these are described in Johnstone and Thorpe, Immunochem-istry in Practice, Second Edition (Blackwell Scientific Pub-lications, 1987).
[0027] As used herein "gene" refers to a nucleic acid segment encoding a protein or transcription product. As used herein, this term can refer to nucleic acid segments contain-ing only a codcontain-ing region, or to nucleic acid segments containing a coding region and any associated expression sequences such as a promoter, a translation initiation sequence, and regulatory sequences. The term gene can refer
to both naturally occurring genes and those produced by genetic manipulation, such as recombinant DNA technology or mutagenesis.
[0028] As used herein "homologous" in reference to genes, other nucleic acid sequences, and proteins, refers to genes, other nucleic acid sequences, and proteins that are similar to each other, respectively. As a practical matter, homologous genes or proteins are identified on the basis of moderate to high sequence identity between the genes or proteins. Homology can also be identified by the presence of highly conserved subsequences in the genes or proteins (even where overall sequence similarity or identity is low). Such conserved subsequences are typically referred to as consensus sequences or domains. As used herein, a "homolog" of a first gene, other nucleic acid sequence, or protein refers to a second gene, other nucleic acid sequence, or protein, respectively, that is homologous to the first gene, other nucleic acid sequence, or protein. Preferred homologs of the poxR gene or the PoxR protein are naturally occurring genes and proteins. Such genes and proteins are preferred targets of mutation in the disclosed method since a preferred purpose of such mutants is to attenuate the natural virulence of the bacteria in which the homologous gene (encoding the homologous protein) is present.
[0029] A preferred means of determining if a gene, other nucleic acid sequence, or protein is homologous to the poxR
gene, a nucleic acid sequence derived from the poxR gene, or the PoxR protein is to determine if the nucleotide or amino acid sequences of the nucleic acids or proteins have a sequence identity with a reference sequence of at least a specified threshold. As used herein, percent nucleotide or amino acid sequence identity is calculated as the percentage of aligned amino acids that match the reference sequence, where the sequence alignment has been determined using the alignment algorithm of Dayhoff et al., Methods in Enzymology 91: 524-545 (1983).
[0030] Preferred homologs of the poxR gene have a nucle-otide sequence identity with nuclenucle-otides 345 to 1319 of SEQ ID NO:1 of greater than 40%, such as at least 50% nucle-otide sequence identity, at least 60% nuclenucle-otide sequence identity, or at least 70% nucleotide sequence identity. Pref-erably, homologs of the poxR gene have at least 80% nucleotide sequence identity; more preferably, at least 90% nucleotide sequence identity; and most preferably, at least 95% nucleotide sequence identity with nucleotides 345 to 1319 of SEQ ID NO:1.
[0031] Other preferred homologs of the poxR gene encode an amino acid sequence having an amino acid sequence identity with SEQ ID NO:2 of greater than 40%, such as at least 50% amino acid sequence identity, at least 60% amino acid sequence identity, or at least 70% amino acid sequence identity. Preferably, homologs of the poxR gene encode an amino acid sequence having at least 80% amino acid sequence identity; more preferably, at least 90% amino acid sequence identity; and most preferably, at least 95% amino acid sequence identity with SEQ ID NO:2.
[0032] Preferred homologs of the PoxR protein have an amino acid sequence identity with SEQ ID NO:2 of greater than 40%, such as at least 50% amino acid sequence identity, at least 60% amino acid sequence identity, or at least 70% amino acid sequence identity. Preferably, homologs of the
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tity; more preferably, at least 90% amino acid sequence diopsis, Streptomyces, Micropolysporas, Thermoactino- identity; and most preferably, at least 95% amino acid mycetes, Mycobacterium, Treponema, Borrelia, Leptospira, sequence identity with SEQ ID NO:2. or Chlamydiae.
[0033] Although poxR gene was identified 15 years ago, molecular and functional data on the gene and gene product are lacking. As disclosed herein, the poxR gene of S. typhimurium was cloned and the nucleotide sequence was determined (Example 1). Comparison of the amino acid sequence derived from the poxR gene to the available databases indicated high homology to an uncharacterized ORF of E. coli 94 min region (91% identity; 96% similarity) and Haemophilus influenzae (65% identity, 79% similarity). The poxR gene product was also characterized (Example 2) and the influence of a poxR mutation on S. typhimurium "pathogenicity island P".
[0034] B. Bacterial Cells
[0035] Any bacterial cells in which a mutation to poxR, or a mutation to a gene in the cell that is homologous to poxR, is suitable for use in the disclosed methods and composition. Preferred bacterial cells are those that colonize, infect, or otherwise grow in or on animals. Particularly preferred are bacterial cells that colonize, infect, or grow in either or both the gastrointestinal tract or respiratory tract. Also preferred are bacterial cells that colonize, infect, or grow in the urogenital tract. Some preferred bacterial cells belong to one of the families Enterobacteriaceae, Micrococcaceae, Vibri-onaceae, Pasteurellaceae, Mycoplasmataceae, or Rickettsi-aceae. Within these families, preferred bacterial cells belong to one of the genera Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micro-coccus, StaphyloMicro-coccus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, or Ehrli-chia. Particular preferred bacterial cells are those that belong to the family Enterobacteriaceae.
[0036] Preferred bacterial cells belong to one of the genera Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Plano-coccus, StomatoPlano-coccus, MicroPlano-coccus, StaphyloPlano-coccus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinoba-cillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus, Entero-coccus, AeroEntero-coccus, Gemella, LactoEntero-coccus, Leuconostoc, Pedicoccus, Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, Rhodococcus, Listeria, Erysipelothrix, Gard-nerella, Neisseria, Camylobacter, Arcobacter, Wolinella, Heliobacter, Achomobacter, Acinetobacter, Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella, Flavi-monas, Flavobacterium, Moraxella, Oligella, PseudoFlavi-monas, Skewanella, Weeksella, Xanthomonas, Bordetella, Francie-sella, Brucella, Legionella, Afipia, Bartonella, Calymmato-bacterium, CardioCalymmato-bacterium, Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus, Bifidobac-terium, EubacBifidobac-terium, Lactobacillus, Rothia, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura, Norcar-
[0037] Particularly preferred are bacterial cells that belong to one of the genera Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, or Morganella. Most preferred are bacterial cells that belong to one of the genera Salmonella or Escherichia. [0038] It is preferred that bacterial cells having a poxR mutation also contain other features which attenuate their virulence and increase their immunogenicity. For example, strains of different Salmonella serotypes can be rendered avirulent by methods known to those skilled in the art, for example, 1) by introducing mutations that impose a require-ment for aromatic amino acids and vitamins derived from precursors in this pathway (Stocker et al., Dev. Biol. Stand. 53:47-54 (1983), Hoiseth et al., Nature 291(5812):238-239 (1981)), 2) by mutating genes for global regulators such as cya and crp (Curtiss and Kelly, Infect. Imm. 55:3035 (1987)), phoP (Miller et al., Proc. Natl. Acad. Sci. USA 86:5054-8 (1989), Galan and Curtiss, Microb. Pathogen. 6:433-443 (1989)), and ompr (Dorman et al., Infect. Immun. 57:2136-40 (1989)), 3) by mutating genes for lipopolysaccharide (LPS) synthesis, such as by galE (Germanier and Furer, Infect. Immun. 4:663-73 (1971), Germanier and Furer, J. Infect. Dis. 131:553-8 (1975)), although this alone may be insufficient (Hone et al., Infect. Immun. 56:1326-1333 (1988)), pmi (Collins et al., Infect. Immun. 59:1079-1085 (1991)), 4) by mutating genes needed for colonization of deep tissues, such as cdt (Kelly et al., Infect. Immun. 60:4881-4890 (1992); Curtiss et al., Devel. Biol. Stand. 82:23-33 (1994)), or 5) by preventing expression of genes for proteases required at high temperature, such as htrA (Johnson et al., Mol. Microbiol. 5:401-407 (1991). Strains possessing mutations in phoQ (Miller et al., Proc. Natl. Acad. Sci. USA 86:5054 (1989)) have the same phenotype as mutations in phoP. Strains with mutations in either phoP or phoQ are referred to herein collectively as phoP mutants. It is preferred that mutations in the above described genes be introduced as deletions since this will preclude reversion mutations and enhance the safety of the strains containing them. Subsequent to the discovery that Salmonella strains with mutations in the genes described above are avirulent and immunogenic, it was observed that many of these strains exhibited, after oral administration, nearly wild-type abili-ties to invade and persist in the GALT and to colonize other lymphoid tissues such as mesenteric lymph nodes, liver, and spleen, but without causing disease symptoms. As a conse-quence, these attenuated strains are capable of stimulating strong mucosal, systemic and cellular immune responses in immunized animal hosts that confer protective immunity to challenge with virulent wild-type Salmonella strains. [0039] Any Salmonella having a poxR mutation can be endowed with the ability to express important colonization or virulence antigens from other bacterial, viral, mycotic and parasitic pathogens at a high level within an immunized animal host (Elements, Pathol. Immunopathol. Res. 6:137-146 (1987); Dougan et al., Parasite Immun. 9:151-60 (1987); Chatfield et al., FEMS Immunol. Med. Microbiol. 7:1-7 (1993); Curtiss et al., in Virulence mechanisms of bacterial pathogens, (Roth, American Society for Microbi-ology, Washington, D.C., 1988) pages 311-328; Curtiss et
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al., Dev. Biol. Stand. 82:23-33 (1994); Doggett and Curtiss, Adv. Exp. Med. Biol. 327:165-73 (1992); Schodel, Sensin Imrnunol. 2:341-9 (1990)).
[0040] C. Method of Producing Bacterial Cells Having Attenuated Virulence
[0041] In S. typhiinurium, mutations in several global regulatory loci including cya/crp (Curtiss and Kelly (1987)), phoPQ (Miller et al. (1989)), rpoS (Robbe-Saule et al., FEMS Microbiol. Lett. 126(2):171-176 (1995)) have been associated with reduced virulence. By examining the role of the poxR gene in the pathogenicity of S. typhimurium, it was discovered that mutation of poxR reduces the virulence of the cells.
[0042] The disclosed method of producing a bacterial cell having attenuated virulence involves mutating the poxR gene, or its homolog, in the bacterial cell. The mutation can be of any type and have any effect on expression of the poxR gene or on the activity of the PoxR protein so long as the virulence of the bacterial cell is reduced. Preferred mutations are those that prevent or reduce transcription of the poxR gene, prevent or reduce translation of poxR mRNA, or which mutate the encoded PoxR protein so that it is no longer active or has reduced activity. In this context, activity of a mutant PoxR protein refers to its phenotypic effect on virulence. Numerous general methods for mutating genes in bacterial cells are known and most are suitable for making poxR mutants. Most preferred are deletion mutations that result in the production of a significantly truncated PoxR protein. Such mutations are stable. An example of the disclosed method involving the production of such a dele-tion mutadele-tion is described in Example 3.
[0043] Deletion mutations of poxR can be introduced into the bacterial chromosome using well known recombinant DNA techniques. For example, the poxR gene can be first cloned onto a suicide vector. The replication of the suicide vector is dependent on a complementing gene on the chro-mosome. When the suicide vector is transferred to a strain lacking the complementing gene, the only means of main-taining the suicide vector is by recombination integrating the suicide vector into the host chromosome. The presence of the poxR gene on the suicide vector results in homologous recombination of the suicide vector into the corresponding gene on the chromosome. This integration will result in a deletion in the gene of chromosomal poxR gene if the wild-type poxR gene on the suicide vector has been altered by deletion of internal regions of the gene prior to integra-tion. This can be accomplished by either restriction enzyme digestion or inverse PCR amplification. The vector-borne poxR gene is thus inactivated while leaving sufficient flank-ing DNA to allow recombination into the chromosome. The defined deletion produced in the suicide vector can be designed to provide a convenient restriction enzyme cloning site allowing the insertion of any foreign gene, such as a gene encoding an antigen. After the initial single recombi-nation event integrating the suicide vector into the chromo-some, a second recombinational event can be selected for by selection against, for example, a tetracycline element on the suicide vector on media containing fusaric acid (Bochner et al., J. Bacteriol. 143:926 (1980)). Alternative mobilizable suicide vectors are also available utilizing sucrose counter selection for the introduction of defined deletions into Sal-monella and other gram negative bacteria (Kaniga et al.,
Gene 109:137-141 (1991)). This results in the replacement of the wild-type allele with the deleted gene containing the desired insert. These methods have been used to produce defined deletions and insertion of foreign genes into the chromosome of Salmonella strains (Chatfield et al., Vaccine 10:53-60 (1992)). A mutation in the poxR gene can also be generated by transformation using linear DNA molecules carrying a selectable marker inserted in poxR.
[0044] It is preferred that the poxR mutation be made by genetic manipulation. As used herein, genetic manipulation refers to a purposeful manipulation of the genome of a bacterial cell directed to altering a specific gene. This can be accomplished, for example, by specifically directing or targeting mutagenesis to a specific gene, or by creating a pool of random mutants and specifically selecting or screen-ing for those in which the poxR gene is mutated. It is intended that genetic manipulation does not encompass genetic changes that occurred naturally, that is, which did not arise through purposeful human intervention or action directed to altering a specific gene. Thus, as used herein, bacterial cells in which the poxR gene has been mutated by genetic manipulation do not include those cells in which the mutation occurred in nature by natural processes.
[0045] Virulence of the disclosed bacterial cells can be assessed by, for example, using the chick model described in Example 6, or the mouse model described in Examples 7 and 8. As used herein, attenuated virulence refers to a reduction in virulence of bacteria relative to a reference bacteria of the same type. It is preferred that the reference bacteria is a wild-type bacteria. Most preferably, the reference bacteria is the parent strain from which a poxR mutant was generated. Avirulent does not mean that a bacterial cell of that genus or species can not ever function as a pathogen, but that the particular bacterial cell being used is avirulent with respect to the particular animal being treated. The bacterial cell may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent. By pathogenic is meant capable of causing disease or impairing normal physiological functioning. Avirulent strains are inca-pable of inducing a full suite of symptoms of the disease that is normally associated with its virulent pathogenic counter-part.
[0046] D. Method of Inducing an Immune Response in an Animal
[0047] The disclosed bacterial cells having attenuated virulence are preferably used to induce an immune response in an animal. As used herein, an immune response refers to the production of antibodies which are immunoreactive to bacterial cells used to induce the response, or to one or more antigens, or nucleic acid encoding an antigen, which are delivered by the bacterial cells. The antibody so produced may belong to any of the immunological classes, such as immunoglobulins A, D, E, G or M. Of particular interest is the production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals. The disclosed bacterial cells are likely to produce a broad range of other immune responses in addition to such IgA formation, for example, cellular and humoral immunity.
[0048] The immune response induced is preferably, but need not be, immunoprotective. As used herein, an immu- noprotective immune response is an immune response which
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reduces the growth, infectivity, pathenogeneity, or viability of an infectious agent in an animal. Most preferred is an immunoprotective immune response which prevents growth, infectivity, pathenogeneity, or viability of an infec-tious agent in an animal.
[0049] The disclosed bacterial cells having a poxR muta-tion can be used to induce an immune response to related bacterial cells (since the disclosed bacterial cells will present their native antigens to a host animal when administered), or, preferably, the disclosed cells can be used as an antigen delivery system. As used herein, the terms antigen delivery system and antigen delivery bacteria refer to bacterial cells that produce an antigen or that harbor a vector encoding an antigen. In one preferred embodiment of an antigen delivery system using the disclosed bacterial cells, pathogenic bac-terial cells that attach to, invade and persist in the gut-associated lymphoid tissue (GALT) or bronchial-gut-associated lymphoid tissue (BALT), and that have a poxR mutation are used as a carrier of a gene product which is used for stimulating immune responses against a pathogen or aller-gen.
[0050] Salmonella having a poxR mutation can be used to protect animals and humans against Salmonella infection. To be useful, such strains need not express any foreign antigen. Salmonella having a poxR mutation and expressing protec-tive antigens from bacterial, viral, mycotic and parasitic pathogens that are efficacious in inducing immunities to protect against infections by these pathogens are preferred for inducing an immune response. Salmonella having a poxR mutation as well as containing other mutations which reduce virulence or which are otherwise of reduced viru-lence are also preferred.
[0051] Shigella or an enteroinvasive E. coli having a poxR mutation can be useful in antigen delivery systems since invasion into colonic mucosa could stimulate lymphoid tissues adjacent to the colon, so as to stimulate a strong mucosal immune response in the reproductive tract. Rectal immunization can be effective because of anatomical fea-tures such as the proximity of lymph nodes and lymphatics to the colon.
[0052] Antigens.
[0053] Live bacteria having a poxR mutation can be used to deliver any product that can be expressed in the bacteria. Preferred expression products for this purpose are antigens. For example, antigens can be from bacterial, viral, mycotic and parasitic pathogens, to protect against bacterial, viral, mycotic, and parasitic infections, respectively; gametes, provided they are gamete specific, to block fertilization; and tumor antigens, to halt cancers. It is specifically contem-plated that antigens from organisms newly identified or newly associated with a disease or pathogenic condition, or new or emerging pathogens of animals or humans, including those now known or identified in the future, can be used with the disclosed bacterial cells and methods. Antigens for use in the disclosed bacterial cells are not limited to those from pathogenic organisms. The selection and recombinant expression of antigens has been previously described by Schodel (1992) and Curtiss (1990). It is preferred that a gene for expression in the disclosed bacterial cells be operably linked to a promoter of any gene of the type III secretion system, preferably a promoter of a sip (ssp) gene, a yop gene, a ipa gene, or a hrp gene. It is also preferred that an
expression product for the disclosed bacterial cells be expressed as a fusion to a Sip (Ssp) protein, a Yop protein, a Ipa protein, or a Hrp protein. Such a fusion is preferably expressed using the natural promoter with which the protein is expressed. Immunogenicity of the bacterial cells can be augmented and/or modulated by constructing strains that also express genes for cytokines, adjuvants, and other immu-nomodulators.
[0054] Some examples are microorganisms useful as a source for antigen are listed below. These include antigens for the control of plague caused by Yersinia pestis and other Yersinia species such as Y. pseudotuberculosis and Y. entero-colitica, of gonorrhea caused by Neisseria gonorrhoea, of syphilis caused by Treponema pallidum, and of venereal diseases as well as eye infections caused by Chlamydia trachomatis. Species of Streptococcus from both group A and group B, such as those species that cause sore throat or heart diseases, Neisseria meningitidis, Mycoplasma pneu-moniae and other Mycoplasma species, Hemophilus influ-enza, Bordetella pertussis, Mycobacterium tuberculosis, Mycobacterium leprae, Bordetella species, Escherichia coli, Streptococcus equi, Streptococcus pneumoniae, Brucella abortus, Pasteurella hemolytica and P. multocida, Vibrio cholera, Shigella species, Borrellia species, Bartonella spe-cies, Heliobacterpylori, Campylobacter spespe-cies, Pseudomo-nas species, Moraxella species, Brucella species, Francisella species, Aeromonas species, Actinobacillus species, Clostridium species, Rickettsia species, Bacillus species, Coxiella species, Ehrlichia species, Listeria species, and Legionella pneumophila are additional examples of bacteria from which antigen genes could be obtained. Viral antigens can also be used with the disclosed bacterial cells. Viral antigens can also be used, including antigens from either DNA or RNA viruses, for example from the classes Papovavirus, Adenovirus, Herpesvirus, Poxvirus, Parvovi-rus, ReoviParvovi-rus, PicornaviParvovi-rus, MyxoviParvovi-rus, ParamyxoviParvovi-rus, or Retrovirus. Antigens of pathogenic fungi, protozoa and parasites can also be used.
[0055] The antigen can also be an allergen of the host such as antigens from pollen and animal dander. Such an antigen can be used in the disclosed bacterial cells in an exposure regimen designed to specifically desensitize an allergic host. [0056] Recombinant Salmonella encoding foreign anti-gens are capable of stimulating strong mucosal, systemic and cellular immune responses against the foreign antigens and thus against the pathogen that is the source of the foreign antigen. It is not necessary that the antigen gene be a complete gene as present in the parent organism, which was capable of producing or regulating the production of a macromolecule, for example, a functioning polypeptide. It is only necessary that the gene be capable of serving as the template used as a guide in the production of an antigenic product. The product may be one that was not found in that exact form in the parent organism. For example, a functional gene coding for a polypeptide antigen comprising 100 amino acid residues may be transferred in part into a carrier bacteria so that a peptide comprising only 75, or even 10, amino acid residues is produced by the cellular mechanism of the host cell. Alternatively, if the amino acid sequence of a particular antigen or fragment thereof is known, it is possible to chemically synthesize the DNA fragment or analog thereof by means of automated gene synthesizers or the like and introduce said DNA sequence into the appro-
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priate expression vector. At the other end of the spectrum is a long section of DNA coding for several gene products, one or all of which can be antigenic. Thus a gene as defined here is any unit of heredity capable of producing an antigen. The gene may be of chromosomal, plasmid, or viral origin. [0057] Multiple antigens can also be expressed by a Sal-monella strain having a poxR mutation. In addition, anti-gens, or even parts of antianti-gens, that constitute a B cell epitope or define a region of an antigen to which an immune response is desired, can be expressed as a fusion to a carrier protein that contains a strong promiscuous T cell epitope and/or serves as an adjuvant and/or facilitates presentation of the antigen to enhance, in all cases, the immune response to the antigen or its component part. This can easily be accomplished by genetically engineering DNA sequences to specify such fusions for expression by the disclosed bacte-rial cells. Fusion to tenus toxin fragment C, CT-B, LT-B and hepatitis virus B core are particularly useful for these purposes, although other epitope presentation systems are well known in the art.
[0058] In order for the disclosed bacteria to be most effective in immunizing an individual, it is preferred that the antigenic material be released in such a way that the immune system of the vaccinated animal can come into play. There-fore the poxR mutant bacteria must be introduced into the animal. In order to stimulate a preferred response of the GALT or BALT cells as discussed previously, introduction of the bacteria or gene product directly into the gut or bronchus is preferred, such as by oral administration, intra-nasal administration, gastric intubation or in the form of aerosols, although other methods of administering the anti-gen delivery bacteria, such as intravenous, intramuscular, subcutaneous injection or intramammary, intrapenial, intrarectal, or vaginal administration, is possible.
[0059] Antigen Delivery Compositions.
[0060] A preferred use of the disclosed bacteria is as vaccines for stimulating an immune response to the deliv-ered antigens. Oral immunization in a suitable animal host with live recombinant Salmonella having a poxR mutation leads to colonization of the gut-associated lymphoid tissue (GALT) or Peyer's patches, which leads to the induction of a generalized mucosal immune response to both Salmonella antigens and any foreign antigens synthesized by the recom-binant Salmonella (Curtiss et al., Adv. Exp. Med. Biol. 251:33-47 (1989)). Further penetration of the bacteria into the mesenteric lymph nodes, liver and spleen augments the induction of systemic and cellular immune responses directed against Salmonella antigens and the foreign anti-gens made by the recombinant Salmonella (Doggett and Curtiss (1992)). Thus the use of recombinant Salmonella for oral immunization stimulates all three branches of the immune system, particularly important when immunizing against infectious disease agents which colonize on and/or invade through mucosal surfaces.
[0061] By vaccine is meant an agent used to stimulate the immune system of a living organism so that an immune response occurs. Preferably, the vaccine is sufficient to stimulate the immune system of a living organism so that protection against future harm is provided. Such protection is referred to herein as immunoprotection. Immunization refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lym-
phocytes can either kill the pathogen and/or activate other cells (for example, phagocytes) to do so in an organism, which is directed against a pathogen or antigen to which the organism has been previously exposed. Although the phrase "immune system" can encompass responses of unicellular organisms to the presence of foreign bodies, as used herein the phrase is restricted to the anatomical features and mechanisms by which a multi-cellular organism responds to an antigenic material which invades the cells of the organism or the extra-cellular fluid of the organism. The antibody so produced may belong to any of the immunological classes, such as immunoglobulins A, D, E, G or M. Of particular interest are recombinant poxR mutant bacteria which stimu-late production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals, although the disclosed bacteria for use as vaccines are not limited to those which stimulate IgA production. For example, bacterial vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example, cellular and humoral immunity. Immune responses to antigens are well studied and widely reported. A survey of immunology is given in Barrett, Textbook of Immunology, Fourth Edition, (C. V. Mosby Co., St. Louis, Mo., 1983), Sites et al., Basic and Clinical Immunology (Lange Medical Books, Los Altos, Calif., 1994), and Orga et al., Handbook of Mucosal Immunology (Academic Press, San Diego, Claif., 1994). Mucosal immunity is also described by McGhee and Mestecky, The Secretory Immune System, Ann. N.Y. Acad. Sci., Volume 409 (1983).
[0062] An individual treated with a vaccine comprising the disclosed bacterial cells is defined herein as including all vertebrates, for example, mammals, including domestic ani-mals and humans, various species of birds, including domes-tic birds, pardomes-ticularly those of agricultural importance. Pref-erably, the individual is a warm-blooded animal.
[0063] The dosages of the disclosed bacterial cells required to elicit an immune response will vary with the antigenicity of the cloned recombinant expression product and need only be a dosage sufficient to induce an immune response typical of existing vaccines. Routine experimenta-tion will easily establish the required dosage. Typical initial dosages of bacterial vaccine for oral administration could be 1x107 to 1x10 11 CFU depending upon the size and age of the individual to be immunized. Administering multiple dosages can also be used as needed to provide the desired level of protective immunity. The pharmaceutical carrier in which the bacterial vaccine is suspended can be any solvent or solid material for encapsulation that is non-toxic to the inoculated animal and compatible with the carrier organism or anti-genic gene product. Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers not used for humans, such as talc or sucrose, or animal feed. Adjuvants may be added to enhance the anti-genicity if desired. When used for administering via the bronchial tubes, the bacterial vaccine is preferably presented in the form of an aerosol.
[0064] Immunization with a pathogen-derived gene prod-uct can also be used in conjunction with prior immunization with a derivative of a pathogenic bacteria which has a poxR mutation and which acts as a carrier to express the pathogen-derived gene product. Such parenteral immunization can
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serve as a booster to enhance expression of the secretory immune response, once the secretory immune system has been primed to that pathogen-derived gene product, by immunization with the recombinant bacteria expressing the pathogen-derived gene product in order to stimulate the lymphoid cells of the GALT or BALT. The enhanced response is known as a secondary, booster, or anamnestic response and results in prolonged immune protection of the host. Booster immunizations may be repeated numerous times with beneficial results.
[0065] Although it is preferred that the disclosed bacterial cells adapted for antigen delivery be administered by routes that stimulate a mucosal immune response, namely oral, intranasal, intravaginal, and interrectal, these bacterial cells can also be delivered intramuscularly, intravenously, and in other parenteral routes. Administration of bacterial cells can also be combined with parenteral administration of purified antigenic components.
[0066] Genetic Immunization.
[0067] The disclosed poxR mutant bacterial cells can also be used to deliver DNA in vivo, and thereby induce an immune response. Delivery of DNA for inducing an immune response is referred to as genetic immunization. For this purpose, it is preferred that such DNA encode an antigen. Preferably, the DNA is in the form of a transfer vector. A bacterial cell having a poxR mutation can harbor a vector for transfer to, and expression in, a cell in an animal or human into which the bacterial cell is placed. As used herein, a transfer vector is an expression vector which can be trans-ferred from a bacterial cell having a poxR mutation into a cell, and which directs the expression of a gene encoded by the transfer vector. It is intended that the transfer vector can contain any gene for expression, including genes encoding antigens, immunomodulators, enzymes, and expression products which regulate gene expression or cellular activity in the recipient cell.
[0068] Preferred recipients for transfer vectors are cells of animal or human hosts. For this purpose, bacterial cells having a poxR mutation and a transfer vector can be administered to an animal host. It is preferred that the bacterial cells invade host cells in order to deliver the transfer vector. For expression of genes on the transfer vector in recipient cells, it is preferred that the genes be operatively linked to expression control sequences operable in the recipient cell. For example, where the recipient cell is an animal or human cell, it is preferred that the genes be operatively linked to a promoter functional in the animal or human.
[0069] Transfer vectors may also contain replication sequences operable in the recipient cell. This would allow replication of the transfer vector, resulting in increased or longer expression of genes present on the transfer vector. Transfer vectors are especially useful for expression of antigens and other proteins that need to be glycosylated or post-translationally modified in a eukaryotic cell. In this way a bacterial cell having a poxR mutation can be used for delivery of a protein requiring eukaryotic processing by expressing the protein from a transfer vector.
[0070] An example of a vector suitable for use as a transfer vector in a bacterial cell having a poxR mutation is described by Sizemore et al., Science 270:299-302 (1995). Sizemore et al. used a construct expressing (3-galactosidase under the control of the immediate early cytomegalovirus promoter and observed the expression of (3-galactosidase in eukaryotic cells following lysis of a Aasd Shigella strain due to DAPless death.
[0071] A preferred use for transfer vectors is in a live bacterial antigen delivery system for stimulation of an immune response in a host animal. For this purpose it is preferred that the bacteria is Salmonella, Shigella, Yersinia, or invasive Escherichia that would invade and then lyse to liberate a transfer vector designed for expression in cells of the animal or human host. This can be useful in stimulating an immune response for viruses, parasites or against gamete antigens where the antigens are normally glycosylated or post-translationally modified in some way that can only be accomplished when the antigen product is synthesized within the eukaryotic cell.
[0072] The efficiency of transfer of a transfer vector or other DNA for genetic immunization can be improved by including an endA mutation, mutations in recBC (with or without sbc suppressor mutations), and/or mutations in other nuclease genes. Such mutations can reduce degradation of the transfer vector or other DNA upon lysis of the bacterial cell. It is also possible to influence the host cell type and the mucosal surface to which the bacterial cell containing the transfer vector or other DNA would adhere to and invade. This can be achieved by blocking or turning on the expres-sion of specific adhesins and/or invasins.
[0073] Many vectors are known for genetic immunization or introduction into cells in an animal or human. Such vectors can be used as transfer vectors in bacterial cells having a poxR mutation. In this case, the bacterial cell having a poxR mutation provides a useful means for intro-ducing such vectors into cells. Preferred promoters for expression of genes on transfer vectors are adenovirus, herpes virus and cytomegalovirus promoters. Expression of the gene can also be increased by placing a bacterial promoter upstream of the eukaryotic promoter, so that the bacterial strain would already express some of the expres-sion product. This expresexpres-sion product would be liberated upon lysis of the bacterial cell.
EXAMPLES
[0074] The bacterial strains and plasmids used or made in the following examples and their sources are listed in Table 1. Strains were grown in L broth or on L agar (Lennox,
Virology 1:190-206 (1955)) or Antibiotic No. 2 (Difco, Detroit, Mich.). When required, antibiotics were added to the growth medium at the following concentration: Kana-mycin, 50 µg/ml; ampicillin, 100 µg/ml; streptoKana-mycin, 100 ug/ml; tetracycline, 12.5 µg/ml.
US 2002/0090376 Al
Jul. 11, 2002
TABLE 1 Bacterial strains
Strain Relevant genotype Reference/Source
S. typhimurium
SM5401 poxA401::Tn10 Van Dyk et al. (1987) SB164 invF::xylE Kaniga et al., Mol. Microbiol.
13(4):555-568 (1994) SB164N invF::xylE poxR401::Tn10 Example 4
SB227 sipC::xylE Kaniga et al., J. Bacteriol. 177(24):7078-7085 (1995) SB227N sipC.:xylE poxR401::Tn10 Example 4
x3761 wild-type UK-1 Curtiss, laboratory collection MGN-762s AphoPQ23 S. Tinge (unpublished) MGN-791s poxR401::Tn10 Example 1
MGN-816s ApoxR401 Example 1 MGN-824s AphoPQ23 Aasd729::xylE Example 4 MGN-939s ApoxR401 (pMEG-250) Example 1 MGN-1036s ApoxR270 Example 3 MGN-1055s ApoxR270 Aasd729::xylE Example 4
E. coli
MGN-617 thi thr leu tonA lacY supE Apir K. Roland (unpublished) recA::RP4-2-Tc::Mu (KmR) Aasdl
CC118Xpir araD139 A (ara-leu)7697 AlacX74 de Lorenzo et al., J. Bacteriol. 172(11):6568-6572 (1991) galK AphoA20 galE recAl rpsE
argE (Am) rpoB thi Apir
Example 1
Cloning of the poxR Gene by Genetic Complementation
[0075] Recombinant DNA techniques were performed according to standard procedures (Sambrook et al., Molecu-lar Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989)). Transformation of plasmid DNA into E. coli and Salmonella strains by electroporation was carried out as described by O'Callaghan and Charbit (Mol. Gen. Genet. 223:157-160 (1990)) using an E. coli Gene Pulser apparatus (Bio-Rad Laboratories). poxR401::Tn10 insertion was moved into S. typhimurium using P22HTint transduc-tion as previously described (Galan et al., J. Bacteriol. 17:4338-4349 (1992)).
[0076] poxA401::Tn10 allele (now referred to as poxR401::Tn10) was introduced into wild-type S. typhimu-rium UK-1 strain x3761 by P22 transduction using a P22 lysate grown on S. typhimurium SMS40l, to generate strain MGN-791s. Bacteriophage P22HTint was used for the trans-ductions (Schmeiger, Mol. Gen. Genet. 119:74-88 (1972)). Although poxR mutants grow slower than the wild-type parent (Chang and Cronan (1982)), this differential growth rate does not appear to be a strong phenotype. Unlike the LT2 derivative strains SMS401 which exhibits a hypersen-sitivity to a-ketobutyrate (AKB) (Van Dyk and LaRossa, J. Bacteriol. 165(2):386-392 (1986); Van Dyk et al. (1987)), it was found that S. typhimurium UK-1 poxR401::Tn10 show a marginal sensitivity to AKB. Therefore, a range of bacte-riological media was screened for substantial difference in growth rates between poxR mutants and isogenic wild-type. It was found that all poxR mutant derivatives of S. typh-imurium produced microcolonies on Antibiotic No. 2 agar (AB2), as compared to LB agar media. A fusaric acid resistant derivative of MGN-791s was selected following
Tn10 deletion to generate MGN-816s. Under the same conditions, the parental strain x3761 produced normal size colonies on both AB2 and LB agar plate. The poxR gene was then cloned by virtue of its ability to restore normal growth to a poxR defective strain on AB2 media.
[0077] A genomic library of wild-type S. typhimurium UK-1 strain x3761 in a pUCl9-derived vector pNEB193 was used (obtained from Steve Tinge). This library was introduced into S. typhimurium strain MGN-816s by elec-troporation and selection on AB2 plate containing ampicil-lin. Among approximately 5,000 ampicillin-resistant micro-colonies, one transformant, MGN-939s, exhibited a large colony morphology indicating that strain MGN-939s had acquired a DNA fragment capable of fully complementing the growth defect of the poxR mutant. The plasmid, desig-nated pMEG-250 was purified and introduced into E. coli CC118 Xpir. A partial restriction map of the 2 kilobase (kb) DNA insert in pMEG-250 was established (FIG. 4). To ensure that the complementation was solely due to cloned poxR and not to an unknown secondary mutation acquired during the selection, pMEG-250 was reintroduced into MGN-816s. All transformants were complemented to the large colony phenotype on AB2. This result strongly sug-gests that the 2 kb DNA fragment of UK-1 in pMEG-250 encodes an active PoxR protein. Alternatively, the DNA fragment could also encode a suppressor of poxR, as the complementing pMEG-250 was a high copy plasmid. To address that possibility, a Southern blot analysis was per-formed on Cla I digested genomic DNA from wild-type x3761 and the transposon Tn10 insertion in poxR strain MGN-791s using labelled 2 kb insert from pMEG-250 as the probe. The probe hybridized to a single Cla I fragment in wild-type and to two Cla I fragments inpoxR401::Tn10. The results confirmed that the 2 kb DNA fragment of pMEG 250 encodes the poxR gene.
